REVIEW
published: 20 November 2015
doi: 10.3389/fphar.2015.00276
The Function of Rho-Associated
Kinases ROCK1 and ROCK2
in the Pathogenesis
of Cardiovascular Disease
Svenja Hartmann 1,2,3 , Anne J. Ridley 3 and Susanne Lutz 1,2*
1
Institute of Pharmacology, University Medical Center Göttingen, Georg-August-University Göttingen, Göttingen,
Germany, 2 German Center for Cardiovascular Research, Göttingen, Germany, 3 Randall Division of Cell and Molecular
Biophysics, King’s College London, London, UK
Edited by:
Friederike Cuello,
University Medical Center
Hamburg-Eppendorf, Germany
Reviewed by:
George Osol,
University of Vermont, USA
Gaetano Santulli,
Federico II University, Italy
*Correspondence:
Susanne Lutz
[email protected]
Specialty section:
This article was submitted to
Cardiovascular and Smooth Muscle
Pharmacology,
a section of the journal
Frontiers in Pharmacology
Received: 26 June 2015
Accepted: 03 November 2015
Published: 20 November 2015
Citation:
Hartmann S, Ridley AJ and Lutz S
(2015) The Function
of Rho-Associated Kinases ROCK1
and ROCK2 in the Pathogenesis
of Cardiovascular Disease.
Front. Pharmacol. 6:276.
doi: 10.3389/fphar.2015.00276
Rho-associated kinases ROCK1 and ROCK2 are serine/threonine kinases that are
downstream targets of the small GTPases RhoA, RhoB, and RhoC. ROCKs are involved
in diverse cellular activities including actin cytoskeleton organization, cell adhesion and
motility, proliferation and apoptosis, remodeling of the extracellular matrix and smooth
muscle cell contraction. The role of ROCK1 and ROCK2 has long been considered to
be similar; however, it is now clear that they do not always have the same functions.
Moreover, depending on their subcellular localization, activation, and other environmental
factors, ROCK signaling can have different effects on cellular function. With respect to
the heart, findings in isoform-specific knockout mice argue for a role of ROCK1 and
ROCK2 in the pathogenesis of cardiac fibrosis and cardiac hypertrophy, respectively.
Increased ROCK activity could play a pivotal role in processes leading to cardiovascular
diseases such as hypertension, pulmonary hypertension, angina pectoris, vasospastic
angina, heart failure, and stroke, and thus ROCK activity is a potential new biomarker for
heart disease. Pharmacological ROCK inhibition reduces the enhanced ROCK activity
in patients, accompanied with a measurable improvement in medical condition. In this
review, we focus on recent findings regarding ROCK signaling in the pathogenesis of
cardiovascular disease, with a special focus on differences between ROCK1 and ROCK2
function.
Keywords: Rho-kinase, ROCK, ROCK signaling, cardiovascular disease, heart, inhibitor, hypertrophy, fibrosis
SIMILARITIES AND DIFFERENCES OF ROCK1 AND ROCK2
IN STRUCTURE AND EXPRESSION
The Rho-associated kinases ROCK1 and ROCK2 belong to the family of serine/threonine AGC
kinases named after the best investigated family members protein kinase A (PKA), G, and C. The
structure of ROCK1 and ROCK2, including important regulatory phosphorylation and cleavage sites
can be seen in Figure 1. ROCK1 and ROCK2 share overall 65% identity in their amino acid sequences
(Nakagawa et al., 1996). ROCK1 and ROCK2 consist of 1354 and 1388 amino acids, respectively,
and both contain an N-terminally located kinase domain, a coiled–coiled region followed by a
Rho-binding domain (RBD; Ishizaki et al., 1996). The RBD binds exclusively to the switch I and
switch II regions of GTP-bound active RhoA, RhoB, and RhoC (Shimizu et al., 2003; Dvorsky
et al., 2004). At the C-terminus, there is a split PH-domain containing a cysteine-rich C1 domain
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FIGURE 1 | ROCK structure and modes of regulation. ROCK1 and ROCK2 consist of an N-terminally located kinase domain and a C-terminally located
pleckstrin homology (PH) domain containing a cysteine-rich C1 domain (CRD). The region between the ROCK kinase domain and the PH domain forms a coiled coil
structure, in which the Rho binding domain (RBD) is located. Both are highly homologous and share overall 64% amino acid sequence identity. A splice variant of
ROCK2 contains an insertion of 57 amino acids following the RBD and is called ROCK2m. ROCK1 and ROCK2 can be activated by binding of RhoGTP to the RBD
and through cleavage of ROCK1 by caspase-3 and of ROCK2 by granzyme B and caspase-2. Autophosphorylation of ROCK1 at Ser1333 and of ROCK2 at
Ser1366 reflects the activation status of the kinases. Phosphorylation of ROCK2 at Thr967, Ser1099, Ser1133, or Ser1374 increased its activation status, whereas
phosphorylation of Tyr722 decreases the ability of ROCK2 to bind to RhoA. Interaction of Thr405 of ROCK2 with the N-terminal extension of the ROCK2’s kinase
domain is essential for substrate phosphorylation and kinase domain dimerization.
(Wen et al., 2008). ROCK1 and ROCK2 have the highest
amino acid homology between their kinase domain (92%),
and are most divergent within their coiled-coil domains with
55% homology. Both full length kinases have similar kinase
activity in vitro (Doran et al., 2004), consistent with the high
homology of the kinase domains. No difference in affinity
of the RBD of ROCK1 and ROCK2 for RhoA, RhoB, or
RhoC has been described. The C-terminal PH-C1 tandem of
ROCKs has been reported to play an autoinhibitory role by
sequestering the N-terminal kinase domain and reducing its
kinase activity (Wen et al., 2008). In addition, the PH-C1 tandem
functions cooperatively in binding to membrane bilayers via the
unconventional positively charged surfaces on each domain (Wen
et al., 2008). However, the PH-C1 domains of ROCK1 and ROCK2
seem to have differential binding preferences for membrane
lipids: the PH-C1 domain of ROCK2 was demonstrated to
bind strongly to phosphatidylinositol (3,4,5)-trisphosphate and
phosphatidylinositol (4,5)-bisphosphate, whereas the one of
ROCK1 did not (Yoneda et al., 2005).
This might partly explain why ROCK1 and ROCK2 were
described to have distinct subcellular distributions, which
vary, however, depending on the cell type and method. For
ROCK2 a cytosolic and nuclear localization, association with
the centrosome, and co-localization with actin and vimentin
filaments in different cell types were reported, as well as its
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localization at the intercalated disk and Z-disk of striated muscle
cells (Leung et al., 1995; Matsui et al., 1996; Sin et al., 1998;
Katoh et al., 2001; Kawabata et al., 2004; Ma et al., 2006; Tanaka
et al., 2006; Iizuka et al., 2012). Further studies point to a
role for ROCK2 in the formation of the contractile ring during
cytokinesis, as it accumulates within the cleavage furrow during
late mitosis (Kosako et al., 1999). In contrast to ROCK2, there
is less information on the subcellular localization of ROCK1.
However, it has been reported to have a cytosolic localization,
as well as association with centrosomes, the plasma membrane,
cell–cell contacts, cell adhesion sites, and vesicles (Chevrier et al.,
2002; Glyn et al., 2003; Stroeken et al., 2006; Iizuka et al., 2012).
In addition to potential differences in subcellular localization,
the expression of ROCK1 and ROCK2 varies between different
tissues. In mouse, ROCK1 mRNA is ubiquitously expressed
except in the brain and muscle, whereas ROCK2 mRNA is
expressed abundantly in the brain, muscle, heart, lung, and
placenta (Nakagawa et al., 1996). In a recent review, Julian and
Olson (2014) analyzed the expression of both kinases based on
expressed sequence tags, confirming the higher abundance of
ROCK2 in heart and brain and suggesting a more prominent
expression of ROCK1 in blood cells and the thymus.
In human and mouse skeletal muscle a unique splice variant
of ROCK2 was detected, named ROCK2m. This variant contains
an insertion of 57 amino acids following the RBD, and is
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progressively expressed during myogenic differentiation together
with ROCK2. Low levels of ROCK2m expression were also
consistently detected in heart and skin. Whether ROCK2m is
differentially regulated, or has a different function to ROCK2 is
not clear so far (Pelosi et al., 2007).
2013). Isoform-specific antibodies against both phosphosites, as
described by Chuang et al. (2012, 2013), provide a new tool to
study ROCK activation, which is normally monitored indirectly
through changes in myosin light chain (MLC) phosphorylation.
Specific Mechanisms of ROCK1 Regulation
SIMILARITIES AND DIFFERENCES IN THE
REGULATION OF ROCK1 AND ROCK2
In addition to being activated by Rho subfamily GTPases and
phosphorylation, ROCKs can be activated in other ways that are
isoform-specific.
An early event in apoptosis is activation of caspases, leading to
cleavage of many proteins, including ROCK1. ROCK1 is cleaved
by caspase-3 at a conserved DETD1113/G sequence, thereby
removing the autoinhibitory C-terminal region and thus resulting
in constitutively active ROCK1 (Sebbagh et al., 2001). This leads
to actomyosin-dependent membrane blebbing (Sebbagh et al.,
2001), which contain fragmented DNA (Coleman et al., 2001). In
heart tissues from patients with heart failure the cleaved ROCK1
fragment, as well as active caspase-3 was detectable, but was
almost absent in normal hearts and in an equivalent cohort of
patients with left ventricular assist devices (Chang et al., 2006).
In animal and cell based models, downregulation of ROCK1
decreased cardiomyocyte apoptosis and interestingly caspase3 activation. This could be due to an Akt-dependent positive
feedback loop linking active ROCK1 to caspase-3 activation,
resulting in a further augmentation of apoptosis (Chang et al.,
2006).
The detrimental role of cleaved ROCK1 for the heart was
further confirmed by transgenic overexpression of C-terminally
truncated ROCK1 (ROCK1∆1) in cardiomyocytes (Yang et al.,
2012). These mice showed an increase in cardiac fibrosis, which
was further augmented by angiotensin II treatment resulting in
a fibrotic cardiomyopathy with diastolic dysfunction. ROCK1mediated activation of the transcription factor, serum response
factor, led to upregulation of TGFβ1 and hence activation of
adjacent cardiac fibroblasts, which could explain the observed
fibrosis. However, it is likely that ROCK1-induced cardiomyocyte
death also contributes to cardiac fibrosis (Yang et al., 2012).
Initially, Rnd3 (also called RhoE) was thought to directly
bind to ROCK1, thus inhibiting downstream signaling. However,
recent data suggests that the Rnd3-dependent inhibition is
indirect and mediated by a Rnd3-p190RhoGAP-dependent
inhibition of Rho/ROCK signaling. Rnd3 belongs to the Rnd
subfamily, which are atypical Rho family proteins. In contrast to
other Rho GTPases, Rnd proteins can bind GTP, however, they
are not able to hydrolyze it and are therefore not regulated by the
classical GTP/GDP conformational switch of small GTPases (Riou
et al., 2010). Rnd3 activity itself is inhibited by ROCK1-dependent
phosphorylation on multiple sites, which induces its translocation
from the membrane to the cytosol and reduces its ability to induce
loss of stress fibers (Riento et al., 2003, 2005; Riou et al., 2013).
With respect to the heart, Rnd3 haploinsufficient mice are
predisposed to left ventricular pressure overload and develop
severe heart failure after transverse aortic constriction (TAC;
Yue et al., 2014). Intriguingly, following TAC operation the
cardiomyocyte apoptosis rate was higher compared to control
animals, correlating with an increase in cleaved caspase3. In addition, ROCK activity was increased. Homozygous
General Mechanism of ROCK Activation
Both the N-terminal and the C-terminal regions of ROCKs
affect catalytic activity (Leung et al., 1996; Jacobs et al., 2006;
Yamaguchi et al., 2006) and both ROCKs form head-to-head
homodimers (Jacobs et al., 2006; Yamaguchi et al., 2006). This
dimerization is dependent on an N-terminal extension region
of the kinase domains, the kinase domain itself, as well as
the coiled-coil domain. The active sites of the two kinases are
thereby positioned in the same direction, possibly facilitating
interactions with dimeric substrates. The C-terminal region of
ROCKs, including the split PH domains, acts as an autoinhibitory
domain by binding directly to the kinase interface: deletion of
this part leads to a constitutive activation in vitro and in vivo
(Leung et al., 1996; Ishizaki et al., 1997; Amano et al., 1999; Jacobs
et al., 2006; Yamaguchi et al., 2006). Upon interaction of the RBD
domain with GTP-loaded RhoA, ROCK activity increases through
a mechanism called derepression: the repression of the kinase
domain by the C-terminal region is removed, which subsequently
leads to an active “open” conformation of the kinase domain.
Whether this simple mechanism is supported or further finetuned by (auto)phosphorylation is not fully understood so far.
Similar to other members of the AGC kinase family, ROCK1
and ROCK2 contain a hydrophobic motif near the C-terminus
of the kinase domain. Biochemical and structural studies of
other AGC kinases have shown that this motif, especially when
phosphorylated, promotes the active conformation of these
kinases. ROCKs share a similar hydrophobic motif and for
ROCK2 it has been shown that the threonine at position 405 in
the hydrophobic motif is essential for substrate phosphorylation
and kinase domain dimerization. However, in contrast to other
kinases like Akt, this does not involve phosphorylation of
Thr405 but interaction with the N-terminal extension of the
kinase domain. In addition, the crystal structures of the ROCK
catalytic domains indicate that, unlike many other protein kinases,
phosphorylation of the activation loop of ROCK is not required
for full catalytic activity (Jacobs et al., 2006; Yamaguchi et al.,
2006). However, Polo-like kinase-1 was shown to phosphorylate
ROCK2 at Thr-967, Ser-1099, Ser-1133, or Ser-1374, thereby
acting together with RhoA to activate ROCK2 (Lowery et al.,
2007). In contrast, phosphorylation of Tyr-722 decreases the
ability of ROCK2 to bind to RhoA (Lee et al., 2010), whereas
dephosphorylation increases the binding (Lee and Chang, 2008).
Although it appears that ROCK activity is not dependent on
(auto)phosphorylation, several phospho-sites in both ROCKs
have been described. Of special experimental interest might be
the autophosphorylation of ROCK1 at Ser1333 and of ROCK2
at Ser1366, as both phosphorylations were suggested to reflect
the activation status of these kinases (Chuang et al., 2012,
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with the Na+ /K+ -ATPase inhibitor ouabain. In these experiments
an additional ouabain-induced cleavage of ROCK1 by caspase
3 was described altogether leading to apoptosis (Ark et al.,
2010). Ouabain is used experimentally to study effects of cardiac
glycosides like digoxin but more interestingly was shown to
be an endogenous cardiotonic steroid upregulated in essential
hypertension, heart and kidney failure (Blaustein, 2014).
As mentioned above, the PH domain of ROCK2 binds
to phosphatidylinositol (3,4,5)-trisphosphate (PIP3 ) and
phosphatidylinositol (4,5)-bisphosphate (PIP2 ), whereas the
PH domain of ROCK1 does not (Yoneda et al., 2005). In
contrast, in ROCK1-null bone marrow macrophages an increased
phosphatase and tensin homolog deleted on chromosome 10
(PTEN) cleavage, reduced phosphorylation, stability, and activity
has been demonstrated, which was accompanied by an enhanced
activation of downstream PTEN targets, including AKT, GSK-3β,
and cyclin D1 (Vemula et al., 2010). PTEN dephosphorylates PIP3
and thus counteracts PI3-kinase, which produces PIP3 (Oudit
et al., 2004). Interestingly, it has been shown in neutrophils
that PI3K activity is localized at the leading edge of the cells
during chemotaxis, whereas PTEN is localized at the back (Li
et al., 2005). This process of differential localization of PTEN
and PI3K activity in the same cell contributes to restricted PIP3
levels in specific compartments like the leading edge during
cell migration (Funamoto et al., 2002). Whether this interplay
involves a differential activation of ROCK1 and ROCK2 has to be
elucidated in the future.
Moreover, ROCK2 expression and activity were found to be
modulated by the peripheral clock gene BMAL. Deletion of
BMAL1 from smooth muscle in mice interfered with blood
pressure circadian rhythm and decreased blood pressure, without
affecting the central pacemaker suprachiasmatic nucleus (SCN).
In mesenteric arteries, BMAL1 deletion suppressed the time-ofday-variations in response to agonist-induced vasoconstriction,
associated with decreased myosin phosphorylation. BMAL1 was
found to bind directly to the promoter of ROCK2 in a time-of-daydependent manner, thereby regulating the time-of-day-variation
in ROCK2 activity. However, it is not clear from the study whether
ROCK1 might also be a BMAL1 target gene (Xie et al., 2015).
Rnd3-null mice died in utero at embryonic stage E11 and isolated
hearts from E10.5 showed a severe apoptotic cardiomyopathy
along with elevated ROCK activity. By crossbreeding the
haploinsufficient Rnd3 with the global ROCK1-knockout (KO)
mice, the augmented effects observed after TAC operation
were strongly reduced, thus arguing for a role of Rnd3-ROCK1
signaling in the regulation of cardiomyocyte apoptosis in heart
disease (Yue et al., 2014). Recently, the function of Rnd3 in
the heart was broadened by demonstrating that β2-adrenergic
receptor density is increased in embryonic hearts from Rnd3-null
mice due to an impaired receptor internalization and degradation
in lysosomes. As a consequence, Rnd3-null mice died between
E10.5 and E12.5 with fetal arrhythmias, probably due to β2adrenergic receptor dependent PKA hyperactivity resulting in
severe Ca2+ leakage through destabilized ryanodine receptor
type 2 Ca2+ release channels (Yang et al., 2015). Whether this
involves Rnd3-dependent Rho/ROCK inhibition or is dependent
on a different Rnd3-regulated mechanism is not known.
The interaction of ROCK1 and Rnd3 is counter-regulated
by the phosphoinositide-dependent kinase-1 (PDK1), which
competes directly with Rnd3 for binding to ROCK1. In the
absence of PDK1, negative regulation of Rho/ROCK signaling
by Rnd3 predominates, causing reduced actomyosin contractility
and motility as shown for cancer cells. The binding of PDK1 to
ROCK1 was independent of protein phosphorylation (Pinner and
Sahai, 2008). So far, regulation of ROCK by PDK1 has not been
investigated in the cardiovascular system.
Specific Mechanisms of ROCK2 Regulation
Like ROCK1, ROCK2 is cleaved and activated by proteases.
Natural killer cells and cytotoxic T-cells can induce apoptosis
in target cells by two distinct mechanisms: a caspase-dependent
mechanism and by exocytosis of cytotoxic granules mainly
releasing perforin and the protease granzyme B (Barry and
Bleackley, 2002). Sebbagh and colleagues demonstrate that
during incubation of lymphokine-activated killer cells together
with myelogenous leukemia K562 target cells a caspaseindependent cleavage of ROCK2 occurred in K562 cells.
ROCK2 was demonstrated to be cleaved by granzyme B at the
IGLD1131 sequence, which is absent in ROCK1. This cleavage
at the C-terminus resulted in a constitutively active ROCK2
fragment of 130 kDa in size. Subsequently to ROCK2 cleavage,
caspase activation occurred and thus cleavage and activation of
ROCK1 (Sebbagh et al., 2005). Additionally, granzyme B cleaves
components of the extracellular matrix, thereby leading to the
release of growth factors like TGFβ1 (Boivin et al., 2012) and
to vascular wall instability and aneurysm (Chamberlain et al.,
2010). A role for granzyme B in diverse cardiovascular diseases
including myocarditis and atherosclerosis has been discussed
(Saito et al., 2011), however, no link to ROCK2 has so far been
established within this context.
As well as granzyme B, caspase-2 cleaves and activates ROCK2.
Upon thrombin stimulation ROCK2, but not ROCK1, was
upregulated and cleaved by caspase-2 into a 140 kDa fragment
in endothelial cells. This led to an increase in the release of
microparticles (Sapet et al., 2006). The specificity of caspase2 for ROCK2 was later confirmed in endothelial cells treated
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ROCK TARGETS AND THE
CARDIOVASCULAR SYSTEM
Similar to many AGC kinases, both ROCK1 and ROCK2
phosphorylate the consensus sequence Arg/Lys–X–Ser/Thr or
Arg/Lys–X–X–Ser/Thr, but other non-consensus sites have been
identified as well (Kang et al., 2007; Riou et al., 2013). Although
ROCKs share this consensus sequence with other kinases like
PKA and PKG, the target overlap is supposedly minor based
on the opposing functions of ROCKs and the cyclic nucleotideregulated kinases in many tissues. In contrast, ROCKs seem to
share substrates with PKCs, however, in some cases with different
kinetics as reviewed below. The specificity of ROCK substrates
has not been thoroughly analyzed for differences, which makes it
difficult to draw a distinction between the two isoforms. To date,
the only well-characterized specific substrate is Rnd3, which was
shown to be phosphorylated by ROCK1 but not ROCK2 (Riento
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FIGURE 2 | ROCK targets. Rho proteins can be activated by guanine nucleotide exchange factors (GEFs), which are themselves activated by receptor tyrosine
kinases (RTKs), G protein coupled receptors (GPCRs), cytokines and integrins. Rho-GTP subsequently activates ROCK1 and ROCK2 that have a broad range of
substrates and are responsible for diverse cellular responses. CIP-17, kinase C-potentiated phosphatase inhibitor of 17 kDa; ERM, ezrin-radixin-moesin; FHOD1,
formin homology 2 domain-containing 1; GAP, GTPase activating protein; LIMK, LIM-kinase; MLC, myosin II regulatory light chain; MYPT1, myosin phosphatase
target subunit 1; PTEN, phosphatase and tensin homolog deleted on chromosome 10.
et al., 2005). An overview of vascular and cardiac ROCK substrates
described in this review can be seen in Figure 2.
In addition, ROCK phosphorylates protein kinase Cpotentiated phosphatase inhibitor of 17 kDa (CPI-17) at Thr38
(Koyama et al., 2000). Initially, PKC was shown to phosphorylate
CPI-17; however, it can be phosphorylated by several other
kinases, including ROCK. This phosphorylation increases the
phosphatase inhibitory potency of CPI-17 by over 1,000-fold
(reviewed by Eto, 2009). Coordinated phosphorylation of CPI-17
by different kinases is believed to be important for regulating
smooth muscle contraction. For example, α1 -adrenergic receptor
stimulation results in a biphasic phosphorylation of CPI-17
through sequential activation of PKC and ROCK. Following
blockade of Ca2+ release from the sarcoplasmic reticulum by the
ryanodine receptor, the α1 -adrenergic receptor-induced, delayed
and sustained activation of ROCK maintains CPI-17 and MYPT1
phosphorylation, causing tonic smooth muscle contraction
(Dimopoulos et al., 2007). Moreover, the ROCK-dependent
Vascular ROCK Substrates
The best characterized ROCK substrate is the myosin phosphatase
target subunit 1 (MYPT1), a regulatory subunit of protein
phosphatase 1 (PP1), also called myosin-binding subunit (MBS)
of myosin phosphatase. MYPT1 in complex with PP1 and the
small subunit M20 counteracts the activity of MLC kinase
and thus decreases the contraction of smooth muscle cells
by dephosphorylating MLC. Depending on the species, ROCK
phosphorylates MYPT1 at Thr-697, Ser-854 or Thr-855, which
attenuates MLCP activity (Feng et al., 1999; Kawano et al., 1999)
and induces the dissociation of MLCP from myosin II (Velasco
et al., 2002). Thus, ROCK increases net MLC phosphorylation
leading to actomyosin contraction.
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regulation of CPI-17 was further substantiated by analysis of
adult MYPT1 smooth muscle-specific KO mice. It was shown
that the observed enhanced phosphorylation of MLC and
contractile force in isolated mesenteric arteries in response
to vasoconstrictors could be inhibited by ROCK and PKC
inhibitors, both resulting in diminished phosphorylation of
CPI-17 (Qiao et al., 2014). There is also evidence that ROCKdependent CPI-17 regulation plays a role in disease. The vascular
smooth muscle contractile hyperactivity detected in isolated
mesenteric arteries from type 2 diabetes db/db-mice was due to
increased RhoA/ROCK activity and CPI-17 phosphorylation.
This activation could also be mimicked by high glucose in
isolated aortic smooth muscle cells (Xie et al., 2006). Finally,
in the context of smooth muscle regulation, ROCK has been
reported to directly phosphorylate Ser19 of MLC, which is the
same residue phosphorylated by MLCK (Amano et al., 1996;
Kureishi et al., 1997).
ERM (ezrin, radixin, and moesin) proteins are suggested to
be downstream targets of ROCK. ERM proteins are closely
related to each other and are thought to function as general
crosslinkers between the plasma membrane and actin filaments.
The last 34 C-terminal amino acids have been demonstrated to
interact with actin filaments (Pestonjamasp et al., 1995). ROCK
was shown to phosphorylate all three proteins at a threonine
in this region in vitro. This phosphorylation interfered with the
intramolecular and/or intermolecular head-to-tail association of
ERM proteins, which is an important regulatory mechanism of
their function (Matsui et al., 1998). In endothelial cells, ERM
proteins have been shown to coordinate the localization of
leukocyte adhesion molecules in a docking structure and play
a role in barrier function, which is partly dependent on ROCK
activity (Barreiro et al., 2002; Guo et al., 2009; Zhang et al.,
2014). In bovine pulmonary artery endothelial cells the adapter
molecule Na+ /H+ exchanger regulatory factor 2 (NHERF2)
was identified to be essential for ROCK–ERM interaction and
ERM phosphorylation (Boratko and Csortos, 2013). However,
other work suggests that in endothelial cells PKCs are the major
activator of proteins (Adyshev et al., 2011, 2013). Also in smooth
muscle, a role for ROCK-dependent ERM phosphorylation was
established. In isolated arteries, ERM proteins were shown to be
phosphorylated in response to vasoconstrictors involving PKC
and ROCK. Interestingly, it was demonstrated that both kinases
showed different kinetics similar as was described for CPI-17
phosphorylation (Koyama et al., 2000). ERM phosphorylation in
smooth muscle cells led to binding of moesin to EBP50, which
is an adaptor molecule, thereby regulating smooth muscle cell
contractility (Baeyens et al., 2010).
LIM-kinases (LIMK1 and LIMK2) are well-characterized
ROCK substrates, which in turn phosphorylate cofilin. This
phosphorylation inhibits the actin-depolymerizing and actinsevering function of cofilin, and thus leads to an increase in
actin filaments (Maekawa et al., 1999; Bravo-Cordero et al.,
2013). LIMK expression is ubiquitous (Foletta et al., 2004). So
far, LIMKs and cofilins have been mainly linked to neurological
disorders and cancer. In endothelial cells it was demonstrated
that shear stress, VEGF and thrombin induce ROCK-dependent
LIMK activation and subsequent cofilin phosphorylation leading
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to sterol regulatory element binding protein 2 activation,
rearrangements of the actin cytoskeleton, and a decrease in cofilin
oligomerization, respectively (Lin et al., 2003; Gong et al., 2004;
Goyal et al., 2013).
ROCK Substrates in the Heart
In contrast to vascular cells, less is known about the roles of
ROCK activity in cardiac cells and most work has focused on
MLC phosphorylation. In most studies on ROCK-dependent
signaling, the phosphorylation of MYPT1 was detected as a
surrogate for ROCK activity (Okamoto et al., 2013; Yue et al.,
2014). In heart tissue, the ROCK targets MYPT1 and the
75% homologous striated muscle-isoform MYPT2 are both
expressed. Electron microscopy indicates that MYPT2 is located
at, or close to, the Z-line, the A band and mitochondria
(Fujioka et al., 1998; Okamoto et al., 2006). Upon α-adrenergic
receptor stimulation, phosphorylation of MYPT and MLC2 was
increased in adult rat ventricular cardiomyocytes, cardiomyocyte
Ca2+ sensitivity of tension was also augmented and all these
effects could be blocked by ROCK inhibition with Y-27632.
Furthermore, activation of α-adrenergic receptors led to a
ROCK-dependent decrease in the PP1-myofilament association
(Rajashree et al., 2005). This spatial redistribution of PP1 could
lead to changes in the phosphorylation pattern of PP1 substrates
at other compartments than the sarcomere. A well-documented
substrate of PP1 in cardiomyocytes is phospholamban, the
regulator of the sarcoplasmic reticulum calcium ATPase (SERCA;
Nicolaou et al., 2009). Interestingly, in adult rat ventricular
cardiomyocytes phenylephrine was indeed shown to significantly
reduce phospholamban phosphorylation leading to a decline in
relaxation velocity (Jeong et al., 2010). In human atrial tissue,
phenylephrine induced a concentration-dependent, sustained
positive inotropic effect accompanied by an increase in MLC2
phosphorylation. In addition, in some tissues a small increase
in relaxation time could be observed. Disinhibition of MLC
phosphatase by ROCK inhibition partially reduced these effects.
In contrast, in ventricular human tissue no influence of
ROCK on the inotropic effect was observed and the effect of
phenylephrine on MLC2 phosphorylation was not consistent
(Grimm et al., 2005). In a tachypacing dog heart model,
ROCK contributed to the phenylephrine-dependent increase
in myofibrillar Ca2+ sensitivity in failing, but not in healthy
ventricular cardiomyocytes. This was accompanied by a higher
expression of Gαq and RhoA, and an augmented phosphorylation
level of MLC2 in the failing myocardium (Suematsu et al.,
2001). In this context, it is important to mention that the
phosphorylation of MLC2 in the healthy contracting heart is in
steady state. The balance is kept by low activities of the MLC kinase
and phosphatase, and not influenced by β-adrenergic signaling.
Phosphorylation disappears in non-contracting, isolated hearts
and in isolated cardiomyocytes, but can be restored by pacing
(Chang et al., 2015) and α-adrenergic signaling as described
above. This steady state phosphorylation status of MLC2 in
the heart is not equally distributed, as it was demonstrated
that MLC2 phosphorylation occurs in a decreasing gradient
from epi- to endocardium and from apex to mid-ventricle with
fiber-to fiber variations. This gradient is thought to play a
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role in ventricular torsion (Davis et al., 2001; Sheikh et al.,
2012).
Other ROCK targets have been sporadically shown to be
regulated in the heart, like ERM proteins (Zhang et al., 2006).
In adult ventricular cardiomyocytes, phosphorylated ERM
is localized at basal state mainly at intercalated disk regions.
However, upon intracellular acidification, phosphorylated ERM
was additionally detectable along the lateral sarcolemma and in
small intracellular dots, and its overall amount was increased.
This increase in phosphorylation could be partly inhibited by
ROCK inhibition (Darmellah et al., 2009). Another ROCK
target in cardiomyocytes is FHOD3, a diaphanous related
formin, which plays a role in actin filament polymerizations.
ROCK1 interacts with and activates FHOD3 involving its
phosphorylation (Iskratsch et al., 2013). Also, ROCK-dependent
cofilin phosphorylation was shown in adult cardiomyocytes. After
treatment of these cells with adiponectin, an important regulator
of peripheral energy metabolism, cofilin phosphorylation
increased in a RhoA/ROCK-dependent manner. This lead
to an increased vesicular trafficking of lipoprotein lipase to
the surface of cardiomyocytes, suggesting a role of ROCK in
metabolism (Ganguly et al., 2011). The muscle specific isoform
cofilin-2 was shown in a KO mouse model to be important for
development, as newborn KO mice were significantly smaller
and weaker compared to their wild type littermates and died by
day eight. Moreover, by day seven the skeletal muscles showed
severe sarcomeric disruptions, accompanied by filamentous
actin accumulation. The onset of pathology in cofilin-2 KO
mice correlated with the normal developmental loss of cofilin-1
expression within myofibers. Interestingly, no cardiac muscle
degeneration could be observed in the cofilin-2 KO mice,
and high expression of cofilin-1 was found within cardiac
myofibers, suggesting that cofilin-1 may be able to compensate
for cofilin-2 deficiency in cardiac muscle (Agrawal et al., 2012).
The importance of cofilin-2 for the heart was demonstrated
recently. Haploinsufficiency of cofilin-2 in cardiomyocytes
resulted in a dilated phenotype with increased left ventricular
volume, and decreased wall thickness and contractile function.
In these mice, myofibrils showed an abnormal actin pattern,
lack of proper orientation, and a poorly organized sarcomere.
Aggregates of sarcomere proteins occur similar to those observed
in idiopathic dilated cardiomyopathy and in rare nemaline
myopathy (Subramanian et al., 2015).
closure and closure of the ventral body wall. Most ROCK1 KO
mice died soon after birth as a result of cannibalization by the
mother. Altered actin cytoskeletal organization with decreased
MLC phosphorylation was identified as the underlying cause of
these phenotypes (Shimizu et al., 2005). However, the phenotype
of the global ROCK1 KO seems to be dependent on the genetic
C57BL/6 background of the mice. On the FVB background
the phenotype was much stronger: 60% of the mice died in
utero before E9.5 (Zhang et al., 2006). Similarly, the ROCK2
KO embryo–placenta interaction phenotype leading to death
in utero on a C57BL/6 × 129/SvJ background was no longer
present when the mice were bred onto a CD-1 (C57BL/6 × Dba)
background (Duffy et al., 2009). Interestingly, ROCK2 KO mice
on a C57BL/6 background showed, in addition to the placental
dysfunction, a failure of eyelid and ventral body wall closure
similar to ROCK1 KO mice (Thumkeo et al., 2005). These findings
clearly demonstrate the impact of the genetic background on the
phenotype of ROCK KO mice.
Vascular Models
ROCKs are implicated in regulating blood pressure by influencing
MLC phosphorylation as reviewed above. However, analysis of
mouse models has not provided conclusive evidence for a link
between ROCKs and blood pressure. In global haploinsufficient
ROCK1 and ROCK2 mouse models, no change in basal
blood pressure was observed (Noma et al., 2008). Similarly, in
haploinsufficient ROCK1+/− mice there was no difference in
angiotensin II induced increase in blood pressure compared to
control mice (Rikitake et al., 2005b). There was also no difference
detected in blood pressure of mice with a vascular smooth muscle
cell (VSMC)-specific, haploinsufficient ROCK2+/− KO under
basal conditions or after 4 weeks in hypoxia. Moreover, in the
same study transgenic mice with almost twofold overexpression
of ROCK2 in smooth muscle cells did not show any changes in
blood pressure under normoxic or hypoxic conditions (Shimizu
et al., 2013). In contrast, in similar haploinsufficient ROCK1+/−
and ROCK2+/− KO mouse lines a decrease in blood pressure
under basal conditions and an attenuation of the diabetes-induced
blood pressure increase was observed (Yao et al., 2013). So far,
the blood pressure has not been determined in mice lacking any
ROCK expression in VSMC.
ROCKs have been implicated in processes after vascular
injury mainly by use of ROCK inhibitors (Shibata et al.,
2001, 2003; Matsumoto et al., 2004). Ligation of the common
carotid artery in mice leads to leukocyte recruitment, neointima
formation, and narrowing of the lumen of the vessel (Schober and
Weber, 2005). Similar changes were observed in haploinsufficient
ROCK2+/− mice. In contrast, in haploinsufficient ROCK1+/−
mice, neointima formation was less pronounced and VSMC
proliferation and survival, as well as expression levels of
proinflammatory adhesion molecules, were decreased. Moreover,
there was reduced leukocyte infiltration. Reciprocal bone marrow
transplantation in wild type and ROCK1 heterozygotes indicated
that leukocytes were primarily responsible for the observed
differences (Noma et al., 2008). This could reflect the important
roles of Rho/ROCK signaling in leukocyte migration, which has
been extensively reviewed elsewhere (Biro et al., 2014).
GENETIC ANIMAL MODELS
OF ROCK1 AND ROCK2
General Knockout of ROCK1 and ROCK2
The first ROCK KO mouse model described was the global KO
of ROCK2 (Thumkeo et al., 2003). Around 90% of ROCK2KO mice died in utero due to a perturbed embryo–placenta
interaction. The surviving animals were significantly smaller after
birth, but subsequently caught up in growth. Histological analyses
of tissues from the surviving adult animals did not reveal any
changes compared to wild type littermates (Thumkeo et al.,
2003). In contrast, the global KO of ROCK1 was less severe
than that of ROCK2. Loss of ROCK1 resulted in failure of eyelid
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There are several lines of evidence that ROCKs play a role in the
pathology of diabetes. ROCK1 KO mice exhibit insulin resistance
and can have a significant increase in glucose-induced insulin
secretion, leading to hyperinsulinemia. Impaired insulin signaling
in skeletal muscle could be responsible for these changes (Lee
et al., 2009). Moreover, it was shown that ROCK1 KO mice treated
with streptozotocin to induce diabetes were protected against the
development of albuminuria, which is a typical sign of kidney
dysfunction in diabetes. This was associated with protection
against kidney fibrosis (Zhou et al., 2011). Under basal conditions
the blood pressure in haploinsufficient ROCK1 or ROCK2 mouse
models was lower compared to control mice and streptozotocininduced increase was absent (Yao et al., 2013). However, there
was no impact on the basal and streptozotocin-induced blood
glucose levels. In diabetes, high glucose levels lead to a decrease
in nitric oxide (NO) synthesis in the vascular endothelium and
hence an impairment of vascular dilatation (Tousoulis et al.,
2013). Accordingly, in aortic rings from diabetic wild type mice,
NO levels were reduced and acetylcholine-dependent dilatation
was impaired. In contrast, aortic rings from haploinsufficient
ROCK1+/− and ROCK2+/− mice produced higher levels of NO
under basal conditions, and a less pronounced difference in
dilatation. Interestingly, reduced ROCK1 expression was more
effective in preventing diabetes-induced changes than ROCK2.
Up-regulation of arginase was reduced in haploinsufficient
diabetic ROCK1 and ROCK2 mice, which could explain the
attenuation of diabetes-induced vascular endothelial dysfunction
and improvement in vascular function during diabetes (Yao
et al., 2013). Studies in macrophages and vascular endothelial
cells have shown that arginase can compete with nitric oxide
synthase (NOS) for -arginine, thereby limiting NO production.
In this context, increased arginase activity has been associated
with vascular dysfunction in diabetes and atherosclerosis (Romero
et al., 2008). Moreover, hyperglycemia was shown to induce
RhoA/ROCK signaling in macrophages, thereby activating JNK
and ERK signaling cascades. This subsequently led to activation
of macrophages and thus eventually to the development of
atherosclerosis (Cheng et al., 2015).
Finally, ROCKs are implicated in the pathogenesis of
pulmonary arterial hypertension (PAH), which is rare but usually
fatal. ROCK2 but not ROCK1 expression is enhanced in the
media of pulmonary arteries and pulmonary arterial smooth
muscle cells from patients with idiopathic PAH (Shimizu et al.,
2013). Conditional ROCK2 KO in VSMC in mice prevented the
chronic hypoxia-induced increase in right ventricular systolic
pressure, as well as the pulmonary vascular remodeling. On
the other hand, transgenic overexpression of ROCK2 in VSMC
augmented both characteristics of PAH. These findings correlated
with changes in mitogenic characteristics in lung tissue including
ERK1/2 phosphorylation. Isolated smooth muscle cells from
the mouse aorta as well as pulmonary arterial smooth muscle
cells from patients showed ROCK2-dependent regulation of cell
proliferation, survival and migration (Shimizu et al., 2013).
modified ROCK1 mice point to a role in cardiac fibrosis.
Cardiac fibrosis is driven by a complex interlinked mechanism
which involves on the one hand the transdifferentiation of
cardiac fibroblasts, endothelial cells, and other cell types into
myofibroblasts, and on the other hand the dysfunction and
apoptosis of cardiomyocytes. As ROCK1 was demonstrated to
propagate apoptosis in cardiomyocytes (Chang et al., 2006), but
also to influence cardiac fibroblast behavior (Haudek et al., 2009),
to date it is not possible to determine whether ROCK1 primarily
functions in cardiomyocytes, cardiac fibroblasts, or both.
Mice with a global ROCK1 deletion that underwent TAC
to induce cardiac hypertrophy had reduced perivascular and
interstitial cardiac fibrosis at 3 weeks, but not at 1 week after
the banding. Expression and secretion of fibrotic cytokines such
as the pro-fibrotic TGFβ2 and connective tissue growth factor
(CTGF), as well as of extracellular matrix proteins was reduced.
Interestingly, ROCK1 deletion did not impair the development
of cardiac hypertrophy. This was supported by findings in
transgenic mice with cardiomyocyte-specific overexpression of
Gαq , which mimics pressure-overload induced hypertrophy, and
homozygous KO of ROCK1. In these mice, the deletion of ROCK1
led to a decreased expression of the fibrogenic factors TGFβ2
and CTGF to the same extent as in hypertrophic hearts of
ROCK1 KO mice (Zhang et al., 2006). In another transgenic
model of Gαq overexpression, ROCK1 deletion attenuated left
ventricular dilation, contractile dysfunction and cardiomyocyte
apoptosis, but did not prevent the development of cardiac
hypertrophy. Interestingly, KO of ROCK1 reduced the induction
of hypertrophic markers, suggesting that ROCK1 might at least be
able to modify cardiac hypertrophy (Shi et al., 2008). Consistent
with this, ROCK1 deficiency might have beneficial effects in
preventing the transition from cardiac hypertrophy to heart
failure. Mice with cardiac overexpression of Gαq develop lethal
cardiomyopathy after pregnancy or at old ages. Interestingly,
deletion of ROCK1 resulted in improved survival, attenuated
left ventricular dilation and wall thinning, and decreased
cardiomyocyte apoptosis. In addition, contractile function was
preserved after pregnancy and in 12-month-old mice. Moreover,
overexpression of ROCK1 in transgenic Gαq mice was associated
with increased cardiomyocyte apoptosis and the development
of decompensated cardiomyopathy, even in mice that were not
subjected to pregnancy stress (Shi et al., 2010).
A role for ROCK1 in cardiac hypertrophy and remodeling was
also addressed using haploinsufficient ROCK1+/− mice, which
were treated either with angiotensin II or NG-nitro--arginine
methyl ester (L-NAME) to inhibit NOS. Both treatments led to a
comparable increase in systemic blood pressure, left ventricular
wall thickness and mass, and hypertrophy in haploinsufficient
ROCK1 and wild type mice. However, similar to ROCK1 KO
mice, the hearts of the haploinsufficient ROCK1 mice showed
less perivascular fibrosis, associated with decreased expression
of TGFβ, CTGF, and collagen III. Likewise, haploinsufficient
ROCK1 mice, which were subjected to TAC or myocardial
infarction showed decreased perivascular fibrosis (Rikitake et al.,
2005b). Transgenic mice expressing a truncated active form of
ROCK1 in cardiomyocytes developed fibrotic cardiomyopathy
and showed elevated NF-κB signaling, as well as SRF-driven
Cardiac Models
So far, no cardiomyocyte or cardiac fibroblast-specific KO
model is available for ROCK1, although data from genetically
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increased TGFβ1 signaling. One reasonable explanation for the
effects on cardiac fibrosis in the different genetic ROCK1 mouse
models is a positive feed-forward regulatory loop of ROCK1 and
caspase-3, as discussed above (Chang et al., 2006; Yang et al.,
2012).
The role of ROCK1 was further studied in a model of
ischemia/reperfusion cardiomyopathy (I/RC) by occlusions of the
left anterior descending artery. ROCK1 KO mice were protected
from the development of I/RC-mediated myocardial dysfunction
and had reduced cardiac fibrosis. This was accompanied by a
lower number of CD34/CD45-positive cells, which are thought to
be blood–borne monocytic fibroblast precursors being attracted
to the myocardium in I/RC (Haudek et al., 2006). Interestingly,
fibroblast formation from human peripheral blood mononuclear
cells was impaired in cells with lower ROCK1 expression
(Haudek et al., 2009). Again, this suggests that ROCK function
in leukocytes plays an important role in ROCK-dependent
responses.
In contrast to the proposed role of ROCK1 in cardiac
fibrosis, ROCK2 was shown to be an important player in cardiac
hypertrophy. Mice with a cardiomyocyte-specific deletion
of ROCK2 display normal cardiac anatomy, function and
hemodynamic parameters under basal conditions. Following
induction of cardiac hypertrophy induced by angiotensin
II infusion or TAC, those mice exhibited substantially less
cardiac hypertrophy, intraventricular fibrosis, cardiac apoptosis,
and oxidative stress compared to control mice. The reduced
hypertrophic phenotype observed in cardiac-specific ROCK2deficient mice may be due to the upregulation of four-and-a-half
LIM-only protein-2 (FHL2). FHL2 was demonstrated to mediate
the inhibition of the transcription factor SRF and extracellular
signal-regulated mitogen-activated protein kinase (ERK), which
are both involved in cardiomyocyte growth. Crossbreeding of
haploinsufficient ROCK2+/− and FHL2 KO mice restored the
hypertrophic response to angiotensin II (Okamoto et al., 2013).
ROCK2 has recently been implicated in metabolic regulation.
After feeding wild type and haploinsufficient ROCK2 mice with a
high fat diet for 17 weeks, it was shown that insulin resistance did
not develop in the haploinsufficient ROCK2 animals. Moreover,
in wild type mice fed with high fat diet, the left ventricular inner
diameter in diastole and the myocardial performance index were
increased, and left ventricular wall motion dyssynchrony was
apparent. None of these changes were present in haploinsufficient
ROCK2 mice after high fat diet. This was associated with
normalization of insulin signaling and GLUT4 expression in the
ROCK2 mouse model (Soliman et al., 2015).
Finally, the complexity of ROCK1 and ROCK2 function in the
heart is nicely demonstrated in an analysis of ROCK1 and ROCK2
expression in two models of cardiac pressure overload: pulmonary
artery constriction (PAC) and TAC to induce overload in the right
(RV) and left ventricle (LV), respectively. PAC induced ROCK2,
but not ROCK1 expression in the first days in the RV, which
was not evident in the LV. In contrast, TAC induced ROCK1 and
ROCK2 expression with a delay of 3 and 7 days, respectively, in
the vascular wall and perivascular area. These changes in ROCK
expression were accompanied by CD45-positive inflammatory
cell infiltration. Transgenic overexpression of a dominant negative
Frontiers in Pharmacology | www.frontiersin.org
Rho-kinase/ROCK2 (DN-RhoK), which inhibits ROCK1 and
ROCK2 function, led to significantly fewer changes in pressureloaded ventricles and improved survival in both PAC and
TAC models. Downregulation of the ERK1/2–GATA4 signaling
pathway in the DN-RhoK-expressing mice could explain its
beneficial effects (Ikeda et al., 2014).
FUNCTION AND REGULATION
OF ROCK1 AND ROCK2 IN HUMAN
CARDIOVASCULAR DISEASE
There are several lines of evidence that ROCK activity is increased
in patients suffering from hypertension, pulmonary hypertension,
stable angina pectoris, vasospastic angina, heart failure, and stroke
(Masumoto et al., 2001, 2002; Shimokawa et al., 2002; Kishi et al.,
2005; Shibuya et al., 2005; Vicari et al., 2005; Ishikura et al., 2006).
An enhanced activity of ROCK signaling was also observed in
smokers (Noma et al., 2005). An overview of the involvement of
ROCK in human cardiovascular disease can be seen in Figure 3.
Where possible, a distinction between ROCK1 and ROCK2 is
made.
A number of compounds have been developed to regulate
ROCK kinase activity. The most widely used are the non-isoformselective Y-27632 and fasudil (also known as HA-1077), which
act as competitive antagonists for ATP in the kinase domain and
do not discriminate between ROCK1 and ROCK2 (Davies et al.,
2000). These inhibitors show a relatively high degree of specificity
for ROCKs, however, when used at higher concentrations they
can also inhibit other kinases, for example protein kinase Crelated kinases (PRKs; Davies et al., 2000), citron kinase (Ishizaki
et al., 2000), or protein kinase A (Ikenoya et al., 2002). Fasudil is
metabolized in the liver to its active metabolite hydroxyfasudil,
which has been reported to exert a more specific inhibitory
effect on ROCKs than fasudil itself (Rikitake et al., 2005a). In
1995, fasudil was approved in Japan and China for prevention
and treatment of cerebral vasospasm following subarachnoid
hemorrhage and is currently the only ROCK inhibitor approved
for human use. In clinical studies, fasudil shows beneficial effects
in patients with PAH, systemic hypertension, vasospastic angina,
stroke, and chronic heart failure (Masumoto et al., 2001, 2002;
Fukumoto et al., 2005; Kishi et al., 2005; Shibuya et al., 2005).
Fasudil was further modified to obtain H-1152P, which is a more
specific inhibitor of ROCKs (Sasaki et al., 2002).
Hypertension is one of the most common cardiovascular
diseases and is characterized by high arterial pressure due to
an increased vascular resistance in the periphery. The reason
for this is often elevated vascular contractility and arterial wall
remodeling, for example as a consequence of atherosclerosis.
Increased activity of the RhoA/ROCK pathway is observed in
experimental hypertension models and hypertensive patients
(Masumoto et al., 2001; Soga et al., 2011; Smith et al., 2013).
For instance, in hypertensive patients treated with the ROCK
inhibitor fasudil the peripheral vascular resistance was decreased
(Masumoto et al., 2001). The underlying mechanism for the
enhanced RhoA/ROCK signaling in hypertension was shown
to be a consequence of the upregulated renin-angiotensinaldosterone system (Seko et al., 2003; Moriki et al., 2004; Wirth
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FIGURE 3 | Role of ROCK1 and ROCK2 in cardiovascular disease. Activated ROCK1 and ROCK2 play a pivotal role in processes leading to cardiovascular
diseases such as hypertension, pulmonary arterial hypertension (PAH), atherosclerosis, vasospastic angina, stroke, diabetes, cardiac ischemia/reperfusion (I/R) injury
and heart failure. Where possible, a distinction between the function of ROCK1 and ROCK2 in the different processes is made.
et al., 2008; Guilluy et al., 2010), and an increased production
of reactive oxygen species (Sun et al., 2008). ROCK inhibitor
treatment, including fasudil, in spontaneously hypertensive rats
(SHR) decreased the mean arterial blood pressure, but not
the systolic blood pressure (Tsounapi et al., 2012). Moreover,
ROCKs were also shown to be involved in structural as well as
functional alterations of blood vessels in SHR (Mukai et al., 2001).
However, ROCK inhibitors do not always lower blood pressure
in hypertension models and, as reviewed above, studies utilizing
ROCK KO mouse models are quite inconclusive. However, there
is strong evidence that ROCK signaling plays an important role in
the pathogenesis of hypertension in humans, especially because it
was shown that polymorphisms in the ROCK2 gene are associated
with a lower risk of developing hypertension (Rankinen et al.,
2008).
Treatment of patients suffering from PAH with the ROCK
inhibitor fasudil induces acute pulmonary vasodilation,
suggesting an involvement of ROCK signaling in PAH (Ishikura
et al., 2006). Interestingly, ROCK2 expression is increased
in the media of pulmonary arteries and pulmonary arterial
smooth muscle cells from patients with idiopathic PAH (Shimizu
et al., 2013). This is supported by findings in a rat model for
PAH. Long-term treatment with fasudil improved pulmonary
hypertension, right ventricular hypertrophy and pulmonary
vascular remodeling, as well as survival. This correlated with
reduced VSMC proliferation concomitant with increased VSMC
Frontiers in Pharmacology | www.frontiersin.org
apoptosis, lower macrophage infiltration, and attenuated VSMC
hypercontractility and endothelial dysfunction (Abe et al., 2004).
Fasudil also appears to improve angina: it increased maximum
exercise time and decreased the number of angina attacks
per week in patients with stable angina (Shimokawa et al.,
2002). Similarly, intracoronary infusion of fasudil attenuated
acetylcholine-induced coronary artery spasm and consequently
myocardial ischemia in patients with vasospastic angina
(Masumoto et al., 2002). These finding are supported by data from
porcine models, in which interleukin 1β was chronically applied
to coronary arteries from the adventitia to induce inflammatory
lesions. Application of fasudil considerably reduced serotonininduced coronary hyperconstriction. Increased activation of the
phosphatase MYPT1, which is normally inhibited by ROCK
phosphorylation, is the likely cause of these effects (Katsumata
et al., 1997; Shimokawa et al., 1999; Kandabashi et al., 2000).
Another clinical study investigated the effects of exogenous NO
on ROCK activity in patients with angina pectoris (Maruhashi
et al., 2015). Although exogenous NO is used intensively in the
clinic as an anti-anginal agent to dilate the vasculature (Abrams,
2005), its long-term effect is still controversial [ISIS-4 (Fourth
International Study of Infarct Survival) Collaborative Group,
1995; Mahmarian et al., 1998; Nakamura et al., 1999]. In this study,
ROCK activity in peripheral leukocytes was decreased in patients
receiving isosorbide mononitrate as NO donor, while ROCK1 and
ROCK2 expression levels were not altered. This indicates that the
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treatment with exogenous NO could have a beneficial effect in
patients with angina pectoris (Maruhashi et al., 2015).
In heart failure patients, administration of fasudil decreased
forearm vascular resistance, accompanied by an enhanced
vasodilation of the forearm, indicating that ROCK might be
involved in the pathogenesis of events leading to heart failure
(Kishi et al., 2005). Indeed, ROCK activity is enhanced in
patients with acute and chronic heart failure. ROCK activity
as measured by the levels of phosphorylated to total MYPT1
in circulating leukocytes (Liu and Liao, 2008) is increased in
patients with chronic heart failure and left ventricular remodeling.
This enhanced activity is accompanied by systolic dysfunction
(Ocaranza et al., 2011). In addition, ROCK activity is increased
in congestive heart failure patients and might be associated
with mortality (Dong et al., 2012). ROCK is also activated in
patients with acute heart failure and seems to decrease during the
time course of the disease. However, no significant correlation
was found between ROCK activity in heart failure patients and
established biomarkers of heart failure, as for instance cardiac
troponin I and brain natriuretic peptide (BNP; Do et al., 2013).
However, ROCK activity is increased in patients with myocardial
infarction, and in combination with high N-terminal pro-B-type
natriuretic peptide could be used as a biomarker to identify
patients suffering from acute coronary syndrome that might be
at high risk (Dong et al., 2013).
Several animal models of heart failure support these findings.
Treatment of hypertensive rats with congestive heart failure with
the ROCK inhibitor Y-27632 attenuated vascular remodeling
and cardiac dysfunction (Kobayashi et al., 2002). Moreover,
mice that underwent myocardial infarction operation and were
treated with fasudil showed improved left ventricular function. In
addition, cardiomyocyte hypertrophy and interstitial fibrosis was
decreased, accompanied by decreased expression of inflammatory
cytokines (Hattori et al., 2004).
There is increasing evidence that inhibition of ROCK leads to
several cardioprotective events in cultured cardiomyocytes from
rat or mouse. For example, ROCK inhibition leads to improved
cardiac contractility due to diminished phosphorylation of
cardiac troponin I/T254, thereby preserving SERCA2a expression
(Vlasblom et al., 2009).
In addition, inhibition of ROCK was shown to inhibit
cardiomyocyte hypertrophy through attenuated angiotensin II
and endothelin-1 signaling (Kuwahara et al., 1999; Yanazume
et al., 2002; Hunter et al., 2009; Ye et al., 2009) and apoptosis via
activation of PI3K/Akt and ERK/MAPK pathways (Chang et al.,
2006; Shi et al., 2010), as well as suppression of the pro-apoptotic
Bcl-2 family protein Bax (Del Re et al., 2007). Moreover, inhibition
of ROCK reduced cardiac fibrosis via the decreased expression of
fibrotic or inflammatory cytokines (Zhang et al., 2006) and greatly
reduces vascular resistance by decreasing MLC phosphorylation
(Kishi et al., 2005).
Taken together, these findings suggest that ROCK activity in
leukocytes may be a new biomarker to predict cardiovascular
events, including arterial hypertension, PAH, angina pectoris,
vasospastic angina and heart failure. This may reflect a crucial
contribution of leukocyte infiltration to the development of
cardiovascular diseases. ROCK is also a potentially valuable
therapeutic target for cardiovascular disease.
CONCLUSIONS AND FUTURE
DIRECTIONS
Since the discovery of ROCKs, a broad range of ROCK substrates
involved in diverse cellular responses has been identified. The
function of both kinases has long been considered to be
similar, however, it has become evident that in addition to
common functions, they also have different targets. Moreover,
depending on their subcellular localization, activation and
other environmental factors, ROCK signaling might lead to
different effects on cellular function. In 2008 the first ROCK2selective inhibitor SLx-2119 was described, which decreases
the expression of CTGF in human dermal fibroblasts and
thus supports a role for ROCK2 in fibrotic processes (Boerma
et al., 2008). This could be a useful inhibitor for dissecting
ROCK2 function, but it will need further characterization to
determine its specificity. However, more selective inhibitors
for ROCK1 versus ROCK2 are needed to allow testing in
vitro and in clinical studies. Indeed, the usage of selective
ROCK inhibitors to treat cardiac diseases could reduce the
side effects that are seen with other inhibitors like fasudil. In
addition, it will be important to further determine selective
functions of ROCK1 and ROCK2, using conditional ROCK1
and ROCK2 depletion in specific tissues or cell types in
mice, e.g., in cardiac fibroblasts or cardiomyocytes. Moreover,
applying the CRISPR/Cas technology to selectively eliminate
each ROCK in human tissue samples would undoubtedly
bring new evidence regarding their distinct signaling. Finally,
there is still an incomplete picture of how and in which
processes ROCKs act as kinases or simply as scaffolding
proteins. In that regard, kinase-dead knock-in mice would
be a useful tool to study ROCK scaffolding functions. To
conclude, there is growing evidence that the ROCK pathway
plays an important role in the development of cardiovascular
diseases and that inhibition of ROCK activity by selective
ROCK inhibitors would be beneficial in treating cardiovascular
diseases.
ACKNOWLEDGMENTS
This work was funded by the German Research Foundation by
projects IRTG1816 RP8 to SH, SFB1002 TPC02 to SL, and by
Cancer Research UK grant C6620/A15961 to AR.
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